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− | <h4 style="text-align: center"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement">TraI | + | <h4 style="text-align: center"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement">TraI Improvement</a></h4> |
<p style="font-size:16px;font-size: 16px; text-indent:1em">At an early stage of our project, we simulated the whole co-culture system using parameters from the C8 production rate of <span style="font-style: italic">E. coli</span>, the iP production rate of human cells and growth inhibition rate of <span style="font-style: italic">mazF</span>. The simulation showed that the C8 production rate is not enough to induce the iP production and as a result, <span style="font-style: italic">E. coli</span> overgrow. | <p style="font-size:16px;font-size: 16px; text-indent:1em">At an early stage of our project, we simulated the whole co-culture system using parameters from the C8 production rate of <span style="font-style: italic">E. coli</span>, the iP production rate of human cells and growth inhibition rate of <span style="font-style: italic">mazF</span>. The simulation showed that the C8 production rate is not enough to induce the iP production and as a result, <span style="font-style: italic">E. coli</span> overgrow. |
Revision as of 11:55, 1 November 2017
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Project Description
Introduction
How can we define a human organism? Is it simply a group of human cells? It's said that in our body, there exist not only 3.0*1013 human cells but also 3.8*1013 bacteria. That means the mass of bacteria reaches 0.2 kg. In other words, humans are not solely composed of human cells. However, in iGEM community, it's been a standard to use single organism in project and it's not an overstatement that most teams don't take it into account that in a real world, multiple kinds of organisms co-exist and the ecosystem is sustained by their mutual dependence. Therefore, to target "true human organism", it's necessary to establish the system that human cells and bacteria co-exist under in vitro conditions. Therefore, we decided to establish co-culture system between human cells and bacteria.
If we can establish a co-culture system, we can find a way to achieve population balance to sustain the co-existence and apply for a medical field like a cancer treatment. If you can co-exist with photosynthetic bacteria or nitrogen fixing bacteria, you can photosynthesize or produce protein from air. If you could co-exist with bacteria, you could be a super human. We named this new type of human 'Coli Sapiens.'
Goal and Approach
Our original goals are as follows:
Establishing an artificial cross-kingdom communication system between human cells and bacteria.
To achieve the first goal, we needed a new cell-to-cell communication system because native and direct communication systems between human cells and bacteria were little known. Thus, we decided to integrate signal transmission system among three kingdoms.
Creating a co-culture model using the cross-kingdom communication and designing ‘Coli Sapiens,’ a new type of human strengthened by bacteria
To achieve the second goal, we chose the essential parts in a complex co-culture system between bacteria and human cells. The reason why co-existence between them has not been developed under in vitro conditions is that a growth rate of bacteria surpasses that of human cells. Thus, when we designed the mathematical model, we emphasized a population of bacteria as one of the biggest factors to establish a co-culture system.
Mechanism
We established the following two systems.
Signal transmission system from bacteria to humans
~ Integration of systems derived from bacteria and humans ~
In our system, the transcription level is controlled by integrating quorum sensing (bacterial cell-to-cell communication) and NF-kB, transcription factor in mammalian cell. We used this system the signal transmission from bacteria to human cells.
![](https://static.igem.org/mediawiki/2017/e/e6/T--TokyoTech--bacteriatohuman.png)
Signal transmission system from human cells to bacteria
~ Integration of systems derived from bacteria and plants ~
In our system, the transcription level is controlled by integrating signal transmission systems derived from bacteria and plants. We used this system the signal transmission from human cells to bacteria.
![](https://static.igem.org/mediawiki/2017/5/5a/T--TokyoTech--humantobacteria.png)
Co-culture system
We conducted experiments to validate the two systems above. Based on the results, we virtually integrated the two systems and conducted population change simulations.
![](https://static.igem.org/mediawiki/2017/a/a9/T--TokyoTech--circuit_map.jpg)
Results
TraI Improvement
At an early stage of our project, we simulated the whole co-culture system using parameters from the C8 production rate of E. coli, the iP production rate of human cells and growth inhibition rate of mazF. The simulation showed that the C8 production rate is not enough to induce the iP production and as a result, E. coli overgrow.
To increase the C8 production rate, we improved the previous genetic circuits in two ways.
- - Introducing various point mutations into CDS of the traI gene and finding a strain whose C8 production rate increases
- - Adding SAM (one of the C8 materials) to culture medium and promoting the C8 production
As a result of the improvement, the concentration of C8 which E. coli produce increased by about 100 folds and it has been possible to induce iP synthesis in human cells from an early stage of E. coli's growth.
![](https://static.igem.org/mediawiki/2017/3/32/T--TokyoTech--TraIimprove50.jpg)
Chimeric Transcription Factor
As for human cells' constructs, we synthesized chimeric transcription factor and iP synthetase genes. In the assay, first, we transduced the constructs. Then, we cultured the cells in which the constructs are successfully transduced and added C8 from E. coli. After the addition, we checked the transcription of atIPT4 and log1 (part of iP synthetase genes) using transcriptome analysis. From this result, we concluded that human cells received C8 from bacteria and successfully produced iP.
![](https://static.igem.org/mediawiki/2017/e/e9/Human_cell_result_v3.png)
The term “Cont” means the control cells that are not electroporated, while “EP” means the electroporated cells. The concentrations of added C8 are indicated below the bars.
AHK4 Assay
We transduced ahk4 into E. coli (KMI002 strain) and cultured them. Then, we added iP and after AHK4 received iP, cps promoter was activated and downstream lacZ is expressed. (lacZ expression was confirmed by blue-white screening.) In conclusion, it turned out that AHK4 can receive iP and induce the gene expression of the downstream genes, which means in a larger scale, E. coli can receive growth inhibition factors from human cells and inhibit the own growth.
![](https://static.igem.org/mediawiki/2017/archive/c/c7/20171028045916%21T--TokyoTech--AHK4qualitive.png)
Cells were grown at room temperature on LB agar plates with and without iP. β-galactosidase activity was monitored by X-gal. Photographs were taken after 25h incubation.
Simulation
We simulated the whole co-culture system again using the assay data. The simulation result showed human cells can control the population of E. coli and the population oscillates.
![](https://static.igem.org/mediawiki/2017/f/f4/T--TokyoTech--oveall.png)
Human Practices
![](https://static.igem.org/mediawiki/2017/f/f8/T--TokyoTech--interaction_complete.png)
From our full year experience in iGEM, we realized the necessity of verifying from a different point of view. In other words, we realized that we researchers ourselves must also continuously reflect on the risks and costs & benefits of the science we discover. In the workshop that we attended as our initial activity in iGEM, we learned from social scientists, the danger of grounding on the deficit model, which fixes on the idea that the general public is ignorant, and the importance of the two-way dialogue between society and researchers.
Hajime Fujita: All Rights Reserved